Abstract: Hearing loss (HL) is the most common sensory disorder affecting more than 28 million Americans. Despite the significant role of genetic factors in the etiology of HL, and astonishing success that has been achieved in the identification of approximately 70 genes for non-syndromic hearing loss (NSHL), much remains to be known about genes involved in the hearing process and the molecular mechanisms of disorders due to defects of these genes. Our long-range goal is to better understand the genetic and molecular basis of hereditary deafness so that effective genetic counseling and successful treatment strategies can be developed. Given the facts that many deafness pedigrees still fail to show linkage to any of the known loci and that mutation frequencies in all the known genes in persons with NSHL remains to be determined, it is therefore important for us to continue identifying new human deafness genes and to complete mutation screening of all known genes for NSHL. The recent technological advances in target- enrichment methods and next generation sequencing (NGS) offers a unique opportunity to break through the barriers of limitations imposed by gene arrays and now allows for the complete analysis of all known deafness-causing genes. The application of NGS will greatly accelerate the pace of disease gene discovery and is now making molecular epidemiological studies of genetic deafness possible for the first time. Interestingly, as shown in our preliminary studies, we have collected a unique group of large families segregating autosomal dominant or recessive deafness, confirmed further heterogeneity of NSHL in these families, successfully identified two new genes using NGS, mapped new loci, and established mutation screening protocol for known NSHL genes. These interesting preliminary results have thus led us to continue identification of novel genes for NSHL and to fully investigate the molecular mechanisms underlying NSHL. Overall, completion of the proposed aims will not only increase our understanding of the biology of hearing and deafness, but will be highly translational by improving the clinical diagnosis of NSHL and patient care. Our Specific Aims in this competitive renewal are: 1. Identify new genes for NSHL. 1a. Identify new genes for autosomal dominant NSHL (ADNSHL) using traditional and innovative technologies; 1b. Identify new genes for autosomal recessive NSHL (ARNSHL) using traditional and innovative technologies in the collected consanguineous families and in the selected probands from deaf x deaf mating families with extensive family histories of NSHL but known not to carry mutations in any known deafness gene. 2. Complete mutation screening of deafness-causing mutations in known NSHL genes. 2a. Determine the prevalence of deafness-causing mutations in known NSHL genes. 2b. Search for clinically relevant genotype- phenotype correlations in our large database.